Phylogenetic relationships among Sarcocystis species in cervids, cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene.

Coccidian parasites in the genus Sarcocystis have a two-host life cycle, and have traditionally been identified on the basis of morphological features of the sarcocyst stage in their intermediate hosts. Additional molecular species identification, delimitation and phylogeny of Sarcocystis spp. have been based mainly on the nuclear ssrRNA gene. This gene is well suited for discrimination between more distant species but less so for closely related species. The objective of this study was therefore to establish the mitochondrial cytochrome c oxidase subunit I gene (cox1) as a novel genetic marker for Sarcocystis spp. and assess its utility for species identification and delimitation. New primers were developed and 1,020-1,095 bp long cox1 sequences were obtained from 155 isolates of 22 Sarcocystis spp. from cattle, sheep, red deer, reindeer, roe deer and moose, and used for phylogenetic reconstructions. For 18 species, the intraspecific and interspecific sequence identities were 98.5-100% and 58-92%, respectively. The four other species had previously been regarded as two species (Sarcocystis rangiferi, Sarcocystis tarandi), each infecting both reindeer and red deer. From cox1 data, each of those appeared to be two separate species, with S. rangiferi and S. tarandi being restricted to reindeer. Thus, cox1 sequences seem to perform better than ssrRNA gene sequences for delimitation of closely related species. The 22 species were distributed in three major clades according to their definitive hosts as in phylogenetic trees obtained from the ssrRNA gene. There were only minor differences in the branching order of different taxa between the trees obtained from either gene. This study has successfully established cox1 as a novel genetic marker for future research on Sarcocystis spp. It has also provided the first published molecular identification of Sarcocystis gigantea and Sarcocystis tenella in Norwegian sheep, and of Sarcocystis hirsuta and Sarcocystis sinensis in Argentinean cattle.